Regulation Of Transgene Expression Research Articles

Vector and helper genome rearrangements occur during production of helper-dependent adenoviral vectors.

Helper-dependent adenoviral vectors (HD Ad) hold extreme promise for gene therapy of human diseases. All viral genes are deleted in HD Ad vectors, and therefore, the presence of a helper virus is required for their production. Current methods to minimize helper contamination in large-scale preparations rely on the use of the Cre/loxP system. The inclusion of loxP sites flanking the packaging signal results in its excision in the presence of Cre recombinase, preventing helper genome encapsidation. It is well established that the level of Cre recombinase activity is important in determining the degree of helper contamination. However, there is little information on other mechanisms that could also play an important role. We have generated several HD Ad vectors containing a rapalog-inducible system to regulate transgene expression, or LacZ under the control of the elongation factor 1 α promoter. Large-scale production of these vectors resulted in abundant helper contamination. Viral DNA analysis revealed the presence of rearrangements between vector and helper genomes. The rearrangements involved a helper DNA molecule with a fragment of the left arm of the HD Ad vector, including its ITR, packaging signal, and some stuffer sequence. Overall, our data suggest that helper DNA molecules that accumulate after Cre recombinase activity are prone to rearrangements, resulting in helper genomes that have incorporated a packaging signal from the vector. Helper particles with rearranged genomes have a growth advantage. This study identifies a novel mechanism leading to helper contamination during helper-dependent adenoviral vector production.

DNA elements reducing transcriptional gene silencing revealed by a novel screening strategy.

Transcriptional gene silencing (TGS)–a phenomenon observed in endogenous genes/transgenes in eukaryotes–is a huge hindrance to transgenic technology and occurs mainly when the genes involved share sequence homology in their promoter regions. TGS depends on chromosomal position, suggesting the existence of genomic elements that suppress TGS. However, no systematic approach to identify such DNA elements has yet been reported. Here, we developed a successful novel screening strategy to identify such elements (anti-silencing regions–ASRs), based on their ability to protect a flanked transgene from TGS. A silenced transgenic tobacco plant in which a subsequently introduced transgene undergoes obligatory promoter-homology dependent TGS in trans allowed the ability of DNA elements to prevent TGS to be used as the screening criterion. We also identified ASRs in a genomic library from a different plant species (Lotus japonicus: a perennial legume); the ASRs include portions of Ty1/copia retrotransposon-like and pararetrovirus-like sequences; the retrotransposon-like sequences also showed interspecies anti-TGS activity in a TGS-induction system in Arabidopsis. Anti-TGS elements could provide effective tools to reduce TGS and ensure proper regulation of transgene expression. Furthermore, the screening strategy described here will also facilitate the efficient identification of new classes of anti-TGS elements.

Adeno-associated Virus-mediated, Mifepristone-regulated Transgene Expression in the Brain

Gene therapy, in its current configuration, is irreversible and does not allow control over transgene expression in case of side effects. Only few regulated vector systems are available, and none of these has reached clinical applicability yet. The mifepristone (Mfp)-regulated Gene Switch (GS) system is characterized by promising features such as being composed of mainly human components and an approved small-molecule drug as an inducer. However, it has not yet been evaluated in adeno-associated virus (AAV) vectors, neither has it been tested for applicability in viral vectors in the central nervous system (CNS). Here, we demonstrate that the GS system can be used successfully in AAV vectors in the brain, and that short-term induced glial cell line-derived neurotrophic factor (GDNF) expression prevented neurodegeneration in a rodent model of Parkinson's disease (PD). We also demonstrate repeated responsiveness to the inducer Mfp and absence of immunological tissue reactions in the rat brain. Human equivalent dosages of Mfp used in this study were lower than those used safely for treatment of psychiatric threats, indicating that the inducer could be safely applied in patients. Our results suggest that the GS system in AAV vectors is well suited for further development towards clinical applicability.

Specific Micro RNA-Regulated TetR-KRAB Transcriptional Control of Transgene Expression in Viral Vector-Transduced Cells

Precise control of transgene expression in a tissue-specific and temporally regulated manner is desirable for many basic and applied investigations gene therapy applications. This is important to regulate dose of transgene products and minimize unwanted effects. Previously described methods have employed tissue specific promoters, miRNA-based transgene silencing or tetR-KRAB-mediated suppression of transgene promoters. To improve on versatility of transgene expression control, we have developed expression systems that use combinations of a tetR-KRAB artificial transgene-repressor, endogenous miRNA silencing machinery and tissue specific promoters. Precise control of transgene expression was demonstrated in liver-, macrophage- and muscle-derived cells. Efficiency was also demonstrated in vivo in murine muscle. This multicomponent and modular regulatory system provides a robust and easily adaptable method for achieving regulated transgene expression in different tissue types. The improved precision of regulation will be useful for many gene therapy applications requiring specific spatiotemporal transgene regulation.

A Hormone Receptor-Based Transactivator Bridges Different Binary Systems to Precisely Control Spatial-Temporal Gene Expression in Drosophila

The GAL4/UAS gene expression system is a precise means of targeted gene expression employed to study biological phenomena in Drosophila. A modified GAL4/UAS system can be conditionally regulated using a temporal and regional gene expression targeting (TARGET) system that responds to heat shock induction. However heat shock-related temperature shifts sometimes cause unexpected physiological responses that confound behavioral analyses. We describe here the construction of a drug-inducible version of this system that takes advantage of tissue-specific GAL4 driver lines to yield either RU486-activated LexA-progesterone receptor chimeras (LexPR) or β-estradiol-activated LexA-estrogen receptor chimeras (XVE). Upon induction, these chimeras bind to a LexA operator (LexAop) and activate transgene expression. Using GFP expression as a marker for induction in fly brain cells, both approaches are capable of tightly and precisely modulating transgene expression in a temporal and dosage-dependent manner. Additionally, tissue-specific GAL4 drivers resulted in target gene expression that was restricted to those specific tissues. Constitutive expression of the active PKA catalytic subunit using these systems altered the sleep pattern of flies, demonstrating that both systems can regulate transgene expression that precisely mimics regulation that was previously engineered using the GeneSwitch/UAS system. Unlike the limited number of GeneSwitch drivers, this approach allows for the usage of the multitudinous, tissue-specific GAL4 lines for studying temporal gene regulation and tissue-specific gene expression. Together, these new inducible systems provide additional, highly valuable tools available to study gene function in Drosophila.

Exploiting Endogenous Micro-RNAs to Avoid off-Target Transgene Expression

Abstract 3296Genetic modification of hematopoietic stem cells (HSCs) has the potential to benefit acquired and congenital hematological disorders. Despite the use of so-called “tissue-specific” promoters to drive expression of the desired transgene, off-target (and consequent deleterious) effects have been observed. MicroRNAs (miRNAs) are important regulators of gene expression. They associate with Argonaute proteins and most typically target 3’UTRs, where complementary base-pairing results in repressed gene expression via RNA decay and translation inhibition. Most miRNAs are ubiquitously expressed, and although some are claimed to be “tissue specific,” such claims have generally not been rigorously validated. The long-term goal of this work is identifying “cell preferential” miRNA expression that could be exploited in expression vectors to minimize off-target transgene expression in HSCs.Initially, total RNA was extracted with Trizol from the megakaryocyte and T-lymphocyte cell lines, Meg-01 and Jurkat, and miRNAs were profiled by Nanostring technology (Nanostring Technologies, Denver, CO). MiR-495 was determined to be highly expressed in Meg-01 and very low in Jurkat cells. A luciferase reporter construct was generated with four canonical binding sites for miR-495 in the 3’UTR and transfected into both cell lines. Compared to control vector without miR-495 binding sites, luciferase expression showed a 50% reduction in Meg-01 cells, but no knock down in Jurkat cells. These experiments indicated that different levels of endogenous miRNA levels can regulate transgene expression through a novel design in the 3’UTR. We next turned our attention to human hematopoietic cells. We reasoned that the long-term goal of minimal off-target transgene expression in HSCs would require knowledge of miRNAs that had little or no detectable expression (“selectively reduced [SR]”) in one cell type and were highly expressed in other cell types. In this manner, the transgene expression would be dampened only in the non-target cells. As a surrogate for bone marrow progenitors and as proof of principle, we used primary cells in normal human peripheral blood. T-cells, B-cells, platelets and granulocytes were purified by density centrifugation followed by immunoselection from five healthy human donors. Flow cytometry using membrane specific markers demonstrate >97% purity of each specific cell preparation. Total RNA was extracted and miRNAs were profiled as above. First, we identified 277 miRNAs that were differentially expressed between any pair of cell types (p-value<0.05 by ANOVA). Second, we performed ranked pair-wise comparisons across all cell types to determine SR miRNAs. This analysis revealed 5 platelet SR-miRNAs, 6 B-cell SR-miRNAs, 2 T-cell SR-miRNAs and 4 granulocyte SR-miRNAs. Lastly, we considered which of these 17 SR-miRNAs would be the best single SR-miRNA within and across cell types. SR-miRNAs were normalized to let-7b, a miRNA we determined to be equivalently expressed across all cell types, and hence, an ideal normalizer. Lineage-specific SR-miRNAs were selected based on extremely low expression in only one cell type and highest fold change of expression compared to the other cell types. The best SR-miRNAs were miR-29b (SR in platelets), miR-125a-5p (SR in B-cells) and miR-146a (SR in granulocytes). The SR expression levels of these 3 miRNAs were validated by qRT-PCR. Our analysis identified no good SR-miRNAs in T-cells. On-going experiments are testing the selective effects of the SR miRNAs in lentiviral vector infection of cord blood CD34+ cells differentiated along specific lineages. In summary, we have demonstrated in hematopoietic cell lines that SR endogenous miRNAs can regulate the expression of transgenes via tandem arrangement of their target sites in the 3’UTR. Additionally, we have identified miRNAs that are specifically expressed at a very low level in one blood cell type and at high levels in other cell types. These miRNAs could potentially be utilized as new biological tools in gene therapy for hematological disorders to restrict transgene expression and avoid the negative consequences of off-target expression. Disclosures:No relevant conflicts of interest to declare.

Further Reduction in Adenovirus Vector-Mediated Liver Transduction without Largely Affecting Transgene Expression in Target Organ by Exploiting MicroRNA-Mediated Regulation and the Cre-loxP Recombination System

In order to detarget undesirable transduction in the liver by an adenovirus (Ad) vector, we previously demonstrated that insertion of sequences perfectly complementary to liver-specific miR-122a into the 3'-untranslated region (UTR) of transgene specifically reduced the transgene expression in the liver by approximately 100-fold; however, a certain level of residual transgene expression was still found in the liver. In order to further suppress the hepatic transduction, we developed a two-Ad vector system that uses the microRNA (miRNA)-regulated transgene expression system and the Cre-loxP recombination system, i.e., insertion of miR-122a target sequences and loxP sites into the transgene expression cassette and coadministration of a Cre recombinase-expressing Ad vector. In addition, to maintain as much as possible the transgene expression in the spleen, which is the target organ of this study, spleen-specific miR-142-3p target sequences were inserted into the 3'-UTR of the Cre recombinase gene to suppress Cre recombinase expression in the spleen. The spleen is an attractive target for immunotherapy because the spleen plays important roles in the immune system. Coadministration of Ad vector possessing CMV promoter-driven Cre recombinase expression cassette with miR-142-3p target sequences resulted in a further 24-fold reduction in the hepatic transgene expression by the Ad vector containing miR-122a target sequences and loxP sites, compared with coadministration of control Ad vector. On the other hand, there was no significant reduction of transgene expression in the spleen.

Destabilizing domains mediate reversible transgene expression in the brain.

Regulating transgene expression in vivo by delivering oral drugs has been a long-time goal for the gene therapy field. A novel gene regulating system based on targeted proteasomal degradation has been recently developed. The system is based on a destabilizing domain (DD) of the Escherichia coli dihydrofolate reductase (DHFR) that directs fused proteins to proteasomal destruction. Creating YFP proteins fused to destabilizing domains enabled TMP based induction of YFP expression in the brain, whereas omission of TMP resulted in loss of YFP expression. Moreover, induction of YFP expression was dose dependent and at higher TMP dosages, induced YFP reached levels comparable to expression of unregulated transgene., Transgene expression could be reversibly regulated using the DD system. Importantly, no adverse effects of TMP treatment or expression of DD-fusion proteins in the brain were observed. To show proof of concept that destabilizing domains derived from DHFR could be used with a biologically active molecule, DD were fused to GDNF, which is a potent neurotrophic factor of dopamine neurons. N-terminal placement of the DD resulted in TMP-regulated release of biologically active GDNF. Our findings suggest that TMP-regulated destabilizing domains can afford transgene regulation in the brain. The fact that GDNF could be regulated is very promising for developing future gene therapies (e.g. for Parkinson's disease) and should be further investigated.

Doxycycline Regulated Induction of AKT in Murine Prostate Drives Proliferation Independently of p27 Cyclin Dependent Kinase Inhibitor Downregulation

The PI3 kinase/AKT pathway has been shown to increase degradation of the p27 cyclin dependent kinase inhibitor through phosphorylation of consensus AKT sites on p27 and SKP2, and AKT driven proliferation may be checked by feedback mechanisms that increase p27 expression and induce senescence. However, these AKT sites are not conserved in mouse, and it has not been clear whether AKT negatively regulates murine p27. Transgenic mice with a probasin promoter controlled prostate specific reverse tetracycline transactivator (ARR2Pb-rtTA) were generated and used to achieve doxycycline inducible expression of a tetracycline operon regulated constitutively active myristoylated AKT1 transgene (tetO-myrAKT). Doxycycline induction of myrAKT occurred within 1 day and rapidly induced proliferation (within 4 days) and the development of prostatic intraepithelial neoplasia (PIN) lesions in ventral prostate, which did not progress to prostate cancer. Cells in these lesions expressed high levels of p27, had increased proliferation, and there was apoptosis of centrally located cells. Doxycycline withdrawal resulted in apoptosis of cells throughout the lesions and rapid clearing of hyperplastic glands, confirming in vivo the critical antiapoptotic functions of AKT. Significantly, analyses of prostates immediately after initiating doxycycline treatment further showed that p27 expression was rapidly increased, coincident with the induction of myrAKT and prior to the development of hyperplasia and PIN. These findings establish in vivo that murine p27 is not negatively regulated by AKT and indicate that proliferation in PI3 kinase/AKT pathway driven mouse models is mediated by p27 independent mechanisms that may be distinct from those in human. Further studies using prostate specific doxycycline regulated transgene expression may be useful to assess the acute effects of inducing additional transgenes in adult murine prostate epithelium, and to assess the requirements for continued transgene expression in transgene induced tumors.

Cloning and function analysis of an alfalfa (Medicago sativa L.) zinc finger protein promoter MsZPP

A 1272 bp upstream sequence of MsZFN gene was cloned from alfalfa, which was designed as MsZPP (Genbank accession number: FJ 161979.2) using an adaptor-mediated genome walking method. A sole transcription start site was located 69 bp upstream of the translation start site. Its pattern of expression included roots, stem vascular tissues, floral reproductive organs, and leaves, but the promoter did not express in seeds, petals or sepals. Transcription levels can be stimulated by dark, MeJA, and IAA. However, GUS fusion activities had no change by treatments of GA, ABA, drought and high salt for 3 days. Deletion analysis revealed that all sections of the promoter can drive gus gene expression in the root, stem, leaves and floral reproductive organs; however, only fragments longer than the -460 bp promoter can stimulate strong gus gene expression in these organs. In addition, the -460 bp promoter fragment can drive gus expression not only in the vascular tissue, but also in leaf guard cells. The results suggest that the promoter MsZPP plays roles in the regulation of transgene expression, particularly due to its darkness, MeJA, and IAA responsiveness.

Hoechst increases adeno‐associated virus‐mediated transgene expression in airway epithelia by inducing the cytomegalovirus promoter

In airway epithelia, the kinetics of recombinant adeno-associated virus (AAV) transgene expression is slow. This has negative practical implications for research, as well as for translation into therapy. The DNA minor groove-binding agent Hoechst-33342 has been shown to enhance AAV transgene expression. In the present study, we investigated the mechanism of Hoechst-related augmentation of AAV-mediated transgene expression. We investigated the effect of Hoechst-33342 on HT1080, COS-7, mouse and human airway epithelia transduced with different AAV serotypes encoding enhanced green fluorescent protein (eGFP). We exposed cells to increasing concentrations of Hoechst-33342 at different time points. We evaluated the effect on second-strand DNA synthesis using AAV with a self-complementary genome. We also investigated the effect on expression from transfected plasmids with and without AAV2 inverted terminal repeats (ITRs). We found that Hoechst-33342 significantly accelerated AAV transgene expression for all serotypes tested. Hoechst-33342 only had an effect when the treatment was given during or after transduction, even 120 days post-transduction, suggesting an effect on transgene expression regulation. Hoechst-33342 increased transgene expression when cells were transduced with a self-complementary AAV with the cytomegalovirus promoter, although there was no effect on cells transduced with conventional single-stranded AAV encoding the Rous sarcoma virus promoter. Finally, Hoechst-33342 increases gene expression from transfected plasmids regardless of the presence of AAV2 ITRs. Hoechst dramatically augments and accelerates AAV-mediated transgene expression in airway epithelia without altering AAV-mediated gene transfer. Hoechst activation of the cytomegalovirus promoter is seen in plasmids, although it is drastically enhanced in the context of AAV.

Efficient in vivo regulation of cytidine deaminase expression in the haematopoietic system using a doxycycline-inducible lentiviral vector system

Regulated transgene expression may reduce transgene-specific and genotoxic risks associated with gene therapy. To prove this concept, we have investigated the suitability of doxycycline (Dox)-inducible human cytidine deaminase (hCDD) overexpression from lentiviral vectors to mediate effective myeloprotection while circumventing the lymphotoxicity observed with constitutive CDD activity. Rapid Dox-mediated transgene induction associated with a 6-17-fold increase in drug resistance was observed in 32D and primary murine bone marrow (BM) cells. Moreover, robust Dox-regulated transgene expression in the entire haematopoietic system was demonstrated for primary and secondary recipients of hCDD-transduced R26-M2rtTA transgenic BM cells. Furthermore, mice were significantly protected from myelosuppressive chemotherapy as evidenced by accelerated recovery of granulocytes (1.9±0.6 vs 1.3±0.3, P=0.034) and platelets (883±194 vs 584±160 10(3) per μl, P=0.011). Minimal transgene expression in the non-induced state and no overt cellular toxicities including lymphotoxicity were detected. Thus, using a relevant murine transplant model our data provide conclusive evidence that drug-resistance transgenes can be expressed in a regulated fashion in the lymphohaematopoietic system, and that Dox-inducible systems may be used to reduce myelotoxic side effect of anticancer chemotherapy or to avoid side effects of high constitutive transgene expression.

505. Physiological Regulation of Transgene Expression by a Lentiviral Vector Containing the A2UCOE Linked to a Myeloid Promoter

505. Physiological Regulation of Transgene Expression by a Lentiviral Vector Containing the A2UCOE Linked to a Myeloid Promoter

Position effect variegation and epigenetic modification of a transgene in a pig model

Sequences proximal to transgene integration sites are able to regulate transgene expression, resulting in complex position effect variegation. Position effect variegation can cause differences in epigenetic modifications, such as DNA methylation and histone acetylation. However, it is not known which factor, position effect or epigenetic modification, plays a more important role in the regulation of transgene expression. We analyzed transgene expression patterns and epigenetic modifications of transgenic pigs expressing green fluorescent protein, driven by the cytomegalovirus (CMV) promoter. DNA hypermethylation and loss of acetylation of specific histone H3 and H4 lysines, except H4K16 acetylation in the CMV promoter, were consistent with a low level of transgene expression. Moreover, the degree of DNA methylation and histone H3/H4 acetylation in the promoter region depended on the integration site; consequently, position effect variegation caused variations in epigenetic modifications. The transgenic pig fibroblast cell lines were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine and/or histone deacetylase inhibitor trichostatin A. Transgene expression was promoted by reversing the DNA hypermethylation and histone hypoacetylation status. The differences in DNA methylation and histone acetylation in the CMV promoter region in these cell lines were not significant; however, significant differences in transgene expression were detected, demonstrating that variegation of transgene expression is affected by the integration site. We conclude that in this pig model, position effect variegation affects transgene expression.

Gene Regulation Systems for Gene Therapy Applications in the Central Nervous System

Substantial progress has been made in the development of novel gene therapy strategies for central nervous system (CNS) disorders in recent years. However, unregulated transgene expression is a significant issue limiting human applications due to the potential side effects from excessive levels of transgenic protein that indiscriminately affect both diseased and nondiseased cells. Gene regulation systems are a tool by which tight tissue-specific and temporal regulation of transgene expression may be achieved. This review covers the features of ideal regulatory systems and summarises the mechanics of current exogenous and endogenous gene regulation systems and their utility in the CNS.

Gene therapy for primary immunodeficiency

Haematopoietic stem cell transplantation (HSCT) is the mainstay of definitive treatment for children with a wide spectrum of primary immunodeficiencies (PIDs), but outcome is heavily dependent on the availability of a human leukocyte antigen-matched donor. Gene therapy using autologous gene-corrected haematopoietic stem cells is an alternative for patients who lack an appropriate donor and has been used to treat children and adults with specific forms of PID, such as severe combined immunodeficiency, for over 10 years. This review summarizes the encouraging long-term outcome data available from these clinical trials and considers the important adverse events that have arisen. Current strategies directed towards improving the efficacy and safety profile of gene therapy will be discussed. Effective clinical trials have been conducted for other forms of PID including chronic granulomatous disease and Wiskott-Aldrich syndrome. Preclinical and clinical studies are now focussed on the development of improved viral vectors giving more regulated or tissue-specific transgene expression with reduced mutagenic potential. Gene therapy offers a valuable alternative management option for selected immunodeficiency patients who lack a suitable donor for HSCT. Clinical trials have confirmed proof-of-principle in terms of stem cell transduction and subsequent immune reconstitution, but have also highlighted the potential for clonal disturbances related to semi-random vector insertion within the genome.

Use of Matrix Attachment Region in Regulating Transgene Expression of Tomato

Use of Matrix Attachment Region in Regulating Transgene Expression of Tomato

Physiological regulation of transgene expression by a lentiviral vector containing the A2UCOE linked to a myeloid promoter

Protection against epigenetic silencing is a desirable feature of future gene therapy vectors, in particular for those applications in which transgene expression will not confer growth advantage to gene-transduced cells. The ubiquitous chromatin opening element (UCOE) consisting of the methylation-free CpG island encompassing the dual divergently transcribed promoters of the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) has been shown to shield constitutive active heterologous promoters from epigenetic modifications and chromosomal position effects. However, it is unclear if this element can be used to improve expression from tissue-specific enhancer/promoters, while maintaining tissue specificity in hematopoietic cells. Here, we evaluated the potential of the A2UCOE in combination with the myeloid-specific myeloid related protein 8 (MRP8) promoter to target transgene expression specifically to myeloid cells in vitro and in vivo from a self-inactivating lentiviral vector. The inclusion of the A2UCOE did not interfere with specific upregulation of MRP8 promoter activity during myeloid differentiation and mediated sustained and vector copy-dependent expression in myeloid cells. Notably, the A2UCOE did not protect the MRP8 promoter from methylation in the P19 embryonal carcinoma cell line, suggesting that this element maintains the inherent epigenetic state and transcriptional activity of cellular promoters in their native configuration. Thus, the A2UCOE could represent a useful protective genetic element in gene therapy vectors, ensuring physiological transcriptional regulation of tissue-specific promoters independent of the chromosomal integration site.

Post-transcriptionally Regulated Expression System in Human Xenogeneic Transplantation Models

Cells have developed a mechanism to discriminate between premature termination codons (PTCs) and normal stop codons during translation, sparking vigorous research to develop drugs promoting readthrough at PTCs to treat genetic disorders caused by PTCs. It was posed that this concept could also be applied to regulated gene therapy protocols by incorporating a PTC into a therapeutic gene, so active protein would only be made after administration of a readthrough agent. The strengths of the system are highlighted here by results demonstrating: (i) background expression levels were reduced to 0.01% to 0.0005% of wild type in unselected mass populations of cells depending upon the specific stop codon utilized and its position within the gene; (ii) expression levels responded well to multiple "On" and "Off" regulation cycles in vivo in human xenograft systems; (iii) the level of induction approached three logs using aminoglycoside activators including NB54, a newly synthesized aminoglycoside with significantly reduced toxicity; and (iv) expression levels could be appreciably altered when employing different promoters in a variety of cell types. These results strongly support the contention that this system should have important clinical applications when tight control of gene expression is required.

MicroRNA-Restricted Transgene Expression in the Retina

BackgroundGene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. Recently, microRNAs have been exploited to fine-tune transgene expression improving therapeutic outcomes. Here we evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions.Methodology/Principal FindingsTo this end, we generated AAV2/5 vectors expressing EGFP and containing four tandem copies of miR-124 or miR-204 complementary sequences in the 3′UTR of the transgene expression cassette. These vectors were administered subretinally to adult C57BL/6 mice and Large White pigs. Our results demonstrate that miR-124 and miR-204 target sequences can efficiently restrict AAV2/5-mediated transgene expression to retinal pigment epithelium and photoreceptors, respectively, in mice and pigs. Interestingly, transgene restriction was observed at low vector doses relevant to therapy.ConclusionsWe conclude that microRNA-mediated regulation of transgene expression can be applied in the retina to either restrict to a specific cell type the robust expression obtained using ubiquitous promoters or to provide an additional layer of gene expression regulation when using cell-specific promoters.